Project Summary/Abstract The steroid hormone estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function, proliferation of breast cancer cells, and concentration of sperm in the male reproductive tract. Estrogens and selective estrogen receptor modulators are widely used in regulating fertility, alleviating postmenopausal symptoms, and preventing and treating breast cancer. The biological effects of these hormones are initiated by binding to the estrogen receptor (ER) and inducing the receptor to bind to estrogen response elements (EREs) residing in target genes. It is this interaction of the receptor with the ERE, in cooperation with multiple coregulatory proteins, that leads to changes in gene expression. During the previous award period, we isolated and identified more than 100 proteins that associate with the DNA-bound ERa and showed that a number of these proteins interact with the receptor, increase the interaction of the receptor with DNA, and influence endogenous, estrogen-responsive gene expression. Interestingly, rather than isolating individual proteins that interact with ERa, we discovered that we had identified interrelated networks of proteins with a variety of enzymatic and catalytic activities that influence basic cellular and physiological processes. A surprising number of these proteins were involved in oxidative stress response and DNA repair, processes that are critical to maintain a normal cell function and have been linked to aging and human diseases. Using molecular, biochemical, cell-based, organotypic brain slice and mouse model systems we will (1) determine whether the recruitment of oxidative stress and DNA repair proteins to ERE-containing DNA alters ERa structure/function and repair of estrogen-responsive genes and (2) delineate whether oxidative stress and DNA repair protein levels are regulated by estrogen and involved in protecting the brain from ischemia. The insights gained will help delineate how estrogens and other hormonal ligands regulate expression of these ERa- associated proteins and enhance our understanding of the cellular responsiveness of the brain to an array of clinically important pharmaceutical agents.